Cryoprotectants for freeze drying of lactic acid bacteria

ABSTRACT

The present invention comprises the discovery and development of an effective cryoprotectant composition, without containing skim milk or any other animal-derived compounds, to achieve long-term stability of freeze-dried lactic acid bacteria (LAB), at different temperatures, whereby the retention of viability of the freeze-dried LAB after 6 months of storage, preferably after 9 months of storage, more preferably after 12 months of storage is more than 50%. The invention is in the field of producing freeze dried bacteria, in particular Lactic acid bacteria. More in particular, the invention relates to the use of a novel combination of cryoprotectants for increasing the viability of bacteria after freeze drying, improving the texture of the lyofilized cake for easy grinding and improving the long term stability of the freeze dried bacteria at different temperature conditions. The invention further relates to such freeze dried bacteria for use in food industry or in human or animal health applications. More in particular, the invention relates to the increased viability and long-term storage of recombinant bacteria capable of expressing heterologous proteins or peptides and administered to humans or animals for therapeutic or vaccination purposes.

The present invention relates to the use of a novel combination ofcryoprotectants for increasing the viability of lactic acid bacteriaafter freeze drying, improving the texture of the lyofilized cake foreasy grinding and improving the long term stability of the freeze driedbacteria at different temperature conditions.

FIELD OF THE INVENTION

The invention is in the field of producing freeze dried bacteria, inparticular Lactic acid bacteria. More in particular, the inventionrelates to the use of a novel combination of cryoprotectants forincreasing the viability of bacteria after freeze drying, improving thetexture of the lyofilized cake for easy grinding and improving the longterm stability of the freeze dried bacteria at different temperatureconditions. The invention further relates to such freeze dried bacteriafor use in food industry or in human or animal health applications. Morein particular, the invention relates to the increased viability andlong-term storage of recombinant bacteria capable of expressingheterologous proteins or peptides and administered to humans or animalsfor therapeutic or vaccination purposes.

BACKGROUND OF THE INVENTION

Lactic acid bacteria (LAB) are a group of taxonomically diverse,Gram-positive bacteria that are able to convert fermentablecarbohydrates mainly into lactic acid, acidifying the growth medium inthe process. In general, LAB species are best known for their use in thefood industry, mainly in the preparation of fermented foods such asdairy products and certain kinds of meat. The commercial significance ofthe dairy fermentation industry, which encompasses production of e.g.cheese, yoghurt and sour cream, is well recognized worldwide.

Over the past decades, interest in LAB has dramatically increased. Thefact that selected LAB strains can influence the intestinal physiologyis widely recognised. L. lactis has enjoyed a growing interest asproduction host for heterologous proteins, and eventually as in situproduction and delivery system for biologically active molecules (seebelow).

At present, much effort has been directed towards the use of geneticallyengineered (GM) LAB species as production and delivery tools fortopical, mucosal administration of biological drugs, includingcytokines, antibody fragments, growth factors, hormones andneuropeptides. (e.g. [6-12]). In particular the engineered food-gradebacterium Lactococcus lactis (L. lactis) was chosen as the preferredmicroorganism for the therapeutic delivery of biologically activepolypeptides. Clearly, the concept of oral therapeutic protein deliveryby engineered L. lactis strains opens exciting possibilities. Anecessary attribute of any pharmaceutical product however is long-termstability (shelf life), typically at least 24 months under predefinedstorage conditions. To this end, an efficient, scalable and reliablemanufacturing platform needs to be developed for engineered L.lactis-based Drug Substance (DS) and Drug Product (DP) formulations.

During manufacturing and subsequent storage, the critical parameter forproduct stability is long-term viability of the engineered bacteria(normally expressed as colony forming units (CFU) per gram in functionof storage time). Manufacture, storage and eventual therapeutic use ofLAB strains imposes significant stress on the bacteria [4]. Onindustrial settings, LAB may be preserved and distributed in liquid,spray-dried, frozen or lyophilized (freeze-dried) forms. While all thesepreparations can be suitable for use as starter cultures in the foodindustry, emphasis is increasingly being placed on long-termpreservation methods that promote high cell viability and metabolicactivity, as these parameters are considered a prerequisite for(bio)pharmaceutical applications. In order to maximize survival,addition of selected cryoprotectants to the biomass and subsequentlyophilization are crucial steps, especially considering the fact thatviable and metabolically active bacteria are an absolute requirement toinduce the desired therapeutic effect in situ.

Freeze-drying is widely regarded as one of the most suitable dehydrationprocesses for bacteria, aiming to achieve a solid and stable finalformulation [4]. It is one of the most common methods to store microbialcell cultures, even though survival rates after freeze-drying and duringstorage may vary between strains [5]. Survival after freeze-dryingreflects the ability of the cells to resist the effects of rapidfreezing and drying, such as membrane lipid oxidation and cell damage atseveral target sites [5]. It is well known that the freeze-drying ofunprotected bacteria kills most of them, and those that survive, dierapidly upon storage. Several attempts have therefore been made toincrease the number of surviving bacteria upon lyophilization andstorage, with limited success (see below).

Lyophilization is by far the most frequently, if not exclusively usedmethod to achieve long-term shelf life [16]. The choice of anappropriate drying medium/cryoprotectant mixture is critical to increasethe survival rate of LAB during lyophilization and subsequent storage[4]. Several studies attempting to increase the survival rate of LABduring freeze-drying and/or subsequent storage have been reported (forreview, see [4]). However, none of these publications demonstratesufficient long-term stability (i.e. >80% survival after one year) ofthe freeze-dried bacteria, as required for pharmaceutical applications,in particular at room temperature (25° C.) or at 2-8° C.

For most LAB cultures of commercial interest for the dairy industry,skim milk powder is selected as drying medium because it stabilizes thecell membrane constituents, facilitates rehydration and forms aprotective coating over the cells [4]. Supplementing skim milk withadditional cryoprotectants agents may enhance its intrinsic protectiveeffect.

Font de Valdez et al. describe the protective effect of adonitol in 10%skim milk, on 12 strains of LAB subjected to freeze-drying [17].Although high survival rates during lyophilization are reported (rangingfrom 42-100%, depending on the strain), no data on long-term stabilitywere provided. Castro et al. assessed the beneficial effects of skimmilk (11%) or trehalose (5%) on the survival of Lactobacillus bulgaricusafter freeze-drying, showing retention rates of 25% (viable cell count)compared to ˜1% in water alone [18]. Again, no data on stability duringsubsequent storage were reported.

Carvalho et al. (2003) demonstrated the stabilizing effect of eithersorbitol or (mono)sodium glutamate (MSG), each added separately to LABsuspended in skim milk, on survival during lyophilization and subsequentstorage for 3-6 months [19]. However, despite the fact that stabilitywas increased compared to skim milk alone, the reported survival ratesin the presence of sorbitol or MSG were still very low (<0.1%).Furthermore, long-term survival of the freeze-dried cells, stored inclosed containers at 20° C. in air and kept in darkness for up to 8months, showed a significant decrease of one or more logs over time.

Carcoba and Rodriguez studied the effects of various compounds, addedindividually to reconstituted skim milk (RSM), on cell survival andmetabolic activity of L. lactis after freeze-drying [16]. They foundthat the sugars trehalose and sucrose, the polyols sorbitol andadonitol, as well as the amino acids β-alanine and glutamic acid, werecapable of enhancing cell viability above the 44.3% recorded in RSMalone. However, actual survival rates with the supplemented media werenot included, and no long-term storage data were disclosed.

As a final example, a study by Huang et al. developed and optimized aprotective medium for Lactobacillus delbrueckii, resulting in a 86% cellviability after freeze-drying [20]. The composition of this medium was:sucrose 66.40 g/L, glycerol 101.20 g/L, sorbitol 113.00 g/L, and skimmilk 130.00 g/L. Again, no long-term stability results were reported.

Huyghebaert et al. aimed to develop a freeze-dried powder formulationcontaining viable GM L. lactis bacteria with an acceptable shelf life[21]. To investigate the influence of the freeze-drying matrix, twodifferent media were used; either M17 broth supplemented with 0.5%glucose (in order to obtain GM17), or 10% (w/v) skim milk supplementedwith 0.5% glucose and 0.5% casein hydrolysate (in order to obtainGC-milk). Following freeze-drying, the influence of lyophilizationparameters, freeze-drying matrix and different storage conditions wasevaluated on short- and long-term viability.

When freeze-dried in conventional GM17 broth, absolute viability wasless than 10%, while freeze-drying in GC-milk matrix resulted insignificantly higher viability (60.0±18.0%). However, despite severalattempts to standardize the freeze-drying procedure, significantbatch-to-batch variability could not be avoided.

Short-term stability studies showed that viability already decreased±20% after freeze-drying and storage for 1 week (GC-milk matrix). Inlong-term stability studies, relative viability was highly decreasedafter 1 month storage, followed by a logarithmic decrease duringsubsequent months of storage (GC-milk matrix, various storageconditions), indicating that long-term stability could not be achieved.

Considering the prior art in its entirety, it is obvious that skim milkis a recurrent component of freeze-drying media for LAB, and thusappears to be essential for bacterial viability. However, the use ofmilk derivatives in novel pharmaceutical compositions is stronglydiscouraged, especially in view of the Transmissible SpongiformEncephalopathy (TSE) risk associated with their use.

Next to high viability after production, freeze-dried LAB should alsohave an acceptable long-term shelf life for pharmaceutical applications.Stabilized dry bacterial compositions are for example described in US2005/0100559, U.S. Pat. No. 3,897,307 and WO2004/065584. In US2005/0100559 the dried bacterial composition are characterized in thatthey comprise a large fraction of stabilizers. See for example [055] inUS 2005/0100559, wherein the stabilizers account for at least 40% (w/v).In WO2004/065584 sucrose or sucrose and maltodextrine were shown toimprove the stability of a bacterial cell culture, but only at −20° C.In this reference there is no indication on how to improve the long-termshelf life (at room temperature) for a composition comprising freezedried bacteria. In U.S. Pat. No. 3,897,307, all experiments start from aculture of different Lactobacillus species in nonfat milk that issubsequently freeze dried, optionally in the presence of stabilizationpotentiators selected from L-ascorbic acid, including edible saltsthereof, and glutamic acid or aspartic acid, including the saltsthereof. As such milk components are an important constituent of thestabilized dry bacterial compositions.

In other words none of the prior art addresses the replacement of themilk components with, sufficient survival and stability under long-termshelf storage. In fact, most of these studies lack precise data oninitial viability, stability and bacterial density. Finally, none ofthem report on freeze-drying of GM bacteria and/or maintenance of theirproperties.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Viable cell count (expressed in Colony Forming Units [CFU]/g) ofL. lactis strain sAGX0037 using different cryoprotectant mixtures: dataimmediately after freeze-drying and after exposure to 25° C./35% RH for24 hours. Detailed composition of the cryoprotectant mixtures isdescribed in Table 1.

FIG. 2: Viable cell count (expressed in Colony Forming Units [CFU]/g) ofL. lactis strain sAGX0037 using different cryoprotectant mixtures: dataimmediately after freeze-drying and after exposure to 25° C./35% RH for24 hours. Detailed composition of the cryoprotectant mixtures isdescribed in Table 3.

FIG. 3: Viable cell count (expressed in Colony Forming Units [CFU]/g) ofL. lactis strain sAGX0037 using cryoprotectant mixture Z4 (20% sodiumglutamate, 10% sorbitol and 10% dextrane 500), freeze-dried at finalshelf temperature of 25° C. and 35° C.: data immediately afterfreeze-drying and after exposure to 25° C./35% RH for 4 and 24 hours.Detailed composition of the bacteria and cryoprotectants mixturecomposition is described in Table 4.

FIG. 4: Stabilizing effect of a combination of sodium glutamate, dextrin(from maize starch) and sorbitol on L. lactis strain sAGX0037 duringfreeze-drying and long-term storage at 3 different storage conditions:−20° C.; 5° C. and 25° C./60% RH in PET/ALU bags. Detailed compositionof the bacteria and cryoprotectants mixture composition is described inTable 5.

DETAILED DESCRIPTION OF THE INVENTION

The present invention comprises the discovery and development of aneffective cryoprotectant composition, without containing skim milk orany other animal-derived compounds, to achieve long-term stability offreeze-dried lactic acid bacteria (LAB), at different temperatures,whereby the retention of viability of the freeze-dried LAB after 6months of storage, preferably after 9 months of storage, more preferablyafter 12 months of storage is more than 50%, preferably more than 60%,more preferably more than 70%, even more preferably more than 80%. Themain advantage of the cryoprotectant composition formulation is toprovide protection for the highly sensitive bacteria duringfreeze-drying, during short-term exposure to normal manufacturingoperational conditions, and during long-term storage after packaging.

The present invention provides a combination of stabilizing agents(cryoprotectants), resulting in high survival of LAB upon freeze-drying,milling and sieving of the freeze-dried cakes and subsequent storage. Inorder to maximize survival, a combination of different stabilizingcompounds (cryoprotectants) is added to the bacterial biomass beforefreeze-drying. This combination of stabilizing compounds, comprising astarch hydrolysate and a glutamic acid salt and/or a polyol, results inimproved survival and stability of freeze-dried LAB. In particular, theinvention relates to the use of a novel combination of cryoprotectantsfor increasing the viability of bacteria after freeze drying, improvingthe texture of the lyophilized cake for easy grinding and improving thelong term stability of the freeze dried bacteria at differenttemperature conditions.

As explained in detail in the examples hereinafter, high viable cellyields (>6×10E+11 colony forming units [CFU]/g) were obtained afterfreeze-drying in the presence of the selected cryoprotectant mixture.Surprisingly, the viability of these freeze-dried cells was not affectedby exposure to environmental conditions (mimicking downstreampharmaceutical formulation and production processes [e.g. capsulefilling]) for 24 hours (25° C./35% RH), and long-term preservation ofcell viability was observed at different storage conditions.

Cryoprotectant combinations containing either sodium glutamate orsorbitol and dextrane, combined with well-known cryoprotectants such astrehalose and sucrose, resulted in viable cell yields immediately afterfreeze-drying that were comparable to the preferred cryoprotectantformulation (code D). Short-term exposure studies clearly demonstratedthat cryoprotectant formulations comprising such combination of a starchhydrolysate and a glutamic acid salt and/or a polyol, protectfreeze-dried L. lactis bacteria upon unprotected storage for 24 hours at25° C. and 35% RH. In the invention, the glutamic acid salt ispreferably a sodium glutamate. The polyol of the invention is preferablysorbitol or mannitol, whereas the starch hydrolysate of the invention ispreferably a dextran.

The results of the survival analysis for Lactococcus lactis (L. lactis)strain sAGX0037, determined by viable cell count on freeze-dried samplesas well as samples exposed to air, indicated that a preferredcombination (code D) of a starch hydrolysate (e.g. dextrane 500), sodiumglutamate and a polyol (e.g. mannitol) (as presented in Examples 1, 2and 3), protected the freeze-dried L. lactis bacteria upon unprotectedstorage for 24 hours at 25° C. and 35% RH.

Compared to a sucrose formulation, which is known as a “golden standard”for stabilisation of freeze-dried LAB, the combination of 3cryoprotectants is clearly superior upon storage, and is the onlycombination of stabilisers that results in viable cell counts >6×10E+11CFU/g upon short-term exposure to 25° C./35% RH. When sodium glutamatealone was added to the bacteria, upon short term exposure, no survivalof the bacteria was observed.

The cryoprotectant combination of a starch hydrolysate (e.g. dextrinfrom maize starch), sodium glutamate and a polyol (e.g. sorbitol), leadsto a stable freeze-dried LAB powder, assuring long-term stability andsurvival of viable bacteria (no significant decrease in viable cellcount was observed on milled and sieved freeze-dried cakes stored at−20° C. and 5° C., and >90% of the initial viable cell count waspreserved during 1 year of storage, resulting in very high CFUconcentrations, up to >5×10E+11 CFU/g). At 25° C., 60% RH, only a slightdecrease in viable cell count was observed, still resulting in high CFUconcentrations, up to >3×10E+11 CFU/g after 1 year of storage at 25°C./60% RH.

It is accordingly an object of the present invention to provide a freezedried bacterial composition comprising the combination of a starchhydrosylate, a glutamic acid salt and a polyl. As is evident from theexamples hereinafter, the amount of starch hydrosylate as used in saidcomposition is from about 2.0% to about 10% (w/v); in particular fromabout 2.5% to about 5% (w/v). The amount of glutamic acid salt as usedin said composition is from about 2.0% to about 10% (w/v); in particularfrom about 5.0% to about 7.5% (w/v). The amount of polyol as used insaid composition is from about 5.0% to about 30% (w/v); in particularfrom about 10% to about 20% (w/v); more in particular from about 7.5% toabout 15% (w/v).

The ‘polyols’ as used herein, generally refers to a mixture of severalsugar alcohols, such as sorbitol, maltitol, and mannitol, amongstothers. It is hydrolyzed from corn starch, potato starch, or wheatstarch, which is broken down into small units such as glucose, dextrin,malto-dextrin, and polydextrin, by amylase enzymes. In a subsequenthydrogenation step, said smaller units are converted into the sugaralcohols, such as sorbitol, maltitol, mannitol, and longer chainhydrogenated saccharides (such as maltitriitol). In a particularembodiment of the present invention the polyol is mannitol, sorbitol ora combination of sorbitol and mannitol. In said embodiment each of saidpolyol, i.e. sorbitol or mannitol, is each independently present in anamount from about 5.0% to about 15% (w/v); in particular from about 7.0%to about 15% (w/v). In an even further embodiment each of said polyolcomponents are present in the same amount, i.e. at about 7%, 8%, 9%,10%, 11%, 12%, 13%, 14% or 15% (w/v).

The ‘glutamic acid salts’ as used herein, generally refers to glutamicacid and its edible water-soluble salts. Such “edible” salts are thoseapproved for use in human foods and are of food grade, such as thesodium and/or potassium salts of glutamic acid. In a particularembodiment of the present invention, the glutamic acid salt ismonosodium glutamate, also known as sodium glutamate and MSG. In an evenfurther embodiment said sodium glutamate is present from about 2.0% toabout 10% (w/v); in particular from about 5.0% to about 7.5% (w/v).

The ‘starch hydrosylates’ as used herein generally refers to thehydrolization products of branched polysaccharides consisting of a largenumber of glucose units, such as starch, or dextran. In starch, thebuilding units consist of the linear and helical amylose, and thebranched amylopectin. In dextran, the straight chain consists of α-1,6glycosidic linkages between glucose molecules, while branches begin fromα-1,4 linkages (and in some cases, α-1,2 and α-1,3 linkages as well). Ina particular embodiment the starch hydrosylates as used herein consistof any one of dextran, 1, dextran, 5, dextran 10, dextran 20, dextran,40, dextran 60, dextran 70, dextran 110, or dextran 500. wherein thenumber refers to the normative molecular weight expressed in kDa; in amore particular embodiment the starch hydrosylate is dextran 500. In aneven further embodiment said dextran is present from about 2.0% to about10% (w/v); in particular from about 2.5% to about 5% (w/v).

As used in the description of the invention and examples, the singularforms “a”, “an”, and “the” include both singular and plural referentsunless the context clearly dictates otherwise. By way of example, “acell” refers to one or more than one cell.

The terms “comprising”, “comprises” and “comprised of” as used hereinare synonymous with “including”, “includes” or “containing”, “contains”,and are inclusive or open-ended and do not exclude additional,non-recited members, elements or method steps.

The recitation of numerical ranges by endpoints includes all numbers andfractions subsumed within that range, as well as the recited endpoints.

The term “about” as used herein when referring to a measurable valuesuch as a parameter, an amount, a temporal duration, and the like, ismeant to encompass variations of +/−20% or less, preferably +/−10% orless, more preferably +/−5% or less, even more preferably +/−1% or less,and still more preferably +/−0.1% or less from the specified value,insofar such variations are appropriate to perform in the disclosedinvention.

All documents cited in the present specification are hereby incorporatedby reference in their entirety. In particular, the teachings of alldocuments herein specifically referred to are incorporated by reference.

Unless otherwise defined, all terms used in disclosing the invention,including technical and scientific terms, have the meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. By means of further guidance, ensuing definitions are includedto better appreciate the teaching of the present invention.

The term “recombinant nucleic acid” refers generally to a nucleic acidwhich is comprised of segments joined together using recombinant DNAtechnology. When a recombinant nucleic replicates in a host organism,the progeny nucleic acids are also encompassed within the term“recombinant nucleic acid”.

Standard reference works setting forth the general principles ofrecombinant DNA technology include Molecular Cloning: A LaboratoryManual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989; Current Protocols inMolecular Biology, ed. Ausubel et al., Greene Publishing andWiley-Interscience, New York, 1992 (with periodic updates) (“Ausubel etal. 1992”); Innis et al., PCR Protocols: A Guide to Methods andApplications, Academic Press: San Diego, 1990. General principles ofmicrobiology are set forth, for example, in Davis, B. D. et al.,Microbiology, 3rd edition, Harper & Row, publishers, Philadelphia, Pa.(1980).

The term “heterologous”, when referring to the relationship between agiven ORF and a promoter, means that the said promoter is not normallyassociated with, i.e., is not normally controlling the transcription of,the said ORF in nature. In other words, the association is created byrecombinant DNA techniques in the recombinant nucleic acids of theinvention.

The term ‘lactic acid bacterium’ generally refers to a bacteriumselected from the group consisting of a Lactococcus species, aLactobacillus species, a Streptococcus species, a Pediococcus species, aBifidobacterium species and a Leuconostoc species and encompasses anytaxon (e.g., species, subspecies, strain) classified as belonging tosuch in the art.

The term “Lactococcus” generally refers to the genus Lactococcus andencompasses any taxon (e.g., species, subspecies, strain) classified asbelonging to such in the art. By means of example, Lactococcus includesthe species Lactococcus garvieae, Lactococcus lactis, Lactococcuspiscium, Lactococcus plantarum and Lactococcus raffinolactis, and anysubspecies and strains thereof.

In preferred embodiments of the invention the Lactococcus is Lactococcuslactis. Lactococcus lactis includes, without limitation, Lactococcuslactis ssp. cremoris, Lactococcus lactis ssp. hordniae, Lactococcuslactis ssp. lactis, Lactococcus lactis ssp. bv. diacetylactis.

In further preferred embodiments of the invention the Lactococcus lactisis Lactococcus lactis ssp. cremoris or Lactococcus lactis ssp. lactis,more preferably Lactococcus lactis ssp. lactis, and encompasses anystrains thereof, such as, e.g., Lactococcus lactis ssp. cremoris SK11 orLactococcus lactis ssp. lactis MG1363.

Accordingly, in an embodiment, the freeze-dried bacterium contains oneor more open reading frames of the recombinant nucleic acids that encodean expression product, preferably a polypeptide, capable of eliciting atherapeutic response in a subject, preferably in a human or animalsubject.

In a particularly useful, exemplary and not limiting, embodiment thesaid one or more open reading frames of the recombinant nucleic acids ofthe invention may encode an antigen and/or a non-vaccinogenictherapeutically active polypeptide.

As used herein, the term “antigen” generally refers to a substanceforeign to a body (esp. to a body of a human or animal subject wheretothe antigen is to be administered) that evokes an immune response,including humoral immunity and/or cellular immunity response, and thatis capable of binding with a product, e.g., an antibody or a T cell, ofthe immune response. Hence, in a preferred example, an antigen requiresa functioning immune system of a subject to which it is administered toelicit a physiological response from such a subject.

An antigen according to the invention may be derived from anypolypeptide to which an immune response in a human or animal subjectwould be therapeutically useful, e.g., from a pathogen, e.g., from aviral, prokaryotic (e.g., bacterial) or eukaryotic pathogen, from anon-physiological protein (e.g., a protein derived from cancer tissue),from allergen (e.g., for eliciting immune tolerance), etc.

The term “a non-vaccinogenic therapeutically active polypeptide” refersgenerally to a polypeptide that, in a human or animal subject to whichit is administered, does not elicit an immune response against itselfand is able to achieve a therapeutic effect. Hence, the therapeuticeffect of such a polypeptide would be expected to be directly linked toits own natural biological function whereby it can achieve particulareffects in a body of a subject; rather than producing a therapeuticeffect by acting as an immunogenic and/or immunoprotective antigen inthe subject. Hence, the non-vaccinogenic therapeutically activepolypeptide should be biologically active in its expressed form or, atleast, must be converted into the biologically active form once releasedfrom the expressing host cell. Preferably, such biologically active formof the said polypeptide may display a secondary and preferably alsotertiary conformation which is the same or closely analogous to itsnative configuration.

Preferably, the non-vaccinogenic therapeutically active polypeptide isalso non-toxic and non-pathogenic.

In a preferred embodiment, the non-vaccinogenic therapeutically activepolypeptide may be derived from human or animal, and may preferablycorrespond to the same taxon as the human or animal subject to which itis to be administered.

Non-limiting examples of suitable non-vaccinogenic therapeuticallyactive polypeptides include ones which are capable of functioninglocally or systemically, e.g., is a polypeptide capable of exertingendocrine activities affecting local or whole-body metabolism and/or thebiologically active polypeptide(s) is/are one(s) which is/are capable ofthe regulation of the activities of cells belonging to theimmunohaemopoeitic system and/or the one or more biologically activepolypeptides is/are one(s) which is/are capable of affecting theviability, growth and differentiation of a variety of normal orneoplastic cells in the body or affecting the immune regulation orinduction of acute phase inflammatory responses to injury and infectionand/or the one or more biologically active polypeptides is/are one(s)which is/are capable of enhancing or inducing resistance to infection ofcells and tissues mediated by chemokines acting on their target cellreceptors, or the proliferation of epithelial cells or the promotion ofwound healing and/or the one or more biologically active polypeptidesmodulates the expression or production of substances by cells in thebody.

Specific examples of such polypeptides include, without limitation,insulin, growth hormone, prolactin, calcitonin, luteinising hormone,parathyroid hormone, somatostatin, thyroid stimulating hormone,vasoactive intestinal polypeptide, cytokines such as IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, any of IL-14 toIL-32, GM-CSF, M-CSF, SCF, IFNs, EPO, G-CSF, LIF, OSM, CNTF, GH, PRL,the TNF family of cytokines, e.g., TNFa, TNFb, CD40, CD27 or FASligands, the IL-1 family of cytokines, the fibroblast growth factorfamily, the platelet derived growth factors, transforming growth factorsand nerve growth factors, the epidermal growth factor family ofcytokines, the insulin related cytokines, etc. Alternatively, thetherapeutically active polypeptide can be a receptor or antagonist forthe therapeutically active polypeptides as defined above. Furtherspecific examples of such suitable polypeptides are listed, e.g., in WO96/11277, page 14, lines 1-30, incorporated herein by reference; in WO97/14806, page 12, line 1 through page 13, line 27, incorporated hereinby reference; or U.S. Pat. No. 5,559,007, col. 8, line 31 through col.9, line 9, incorporated by reference herein.

Accordingly, in an embodiment the recombinant nucleic acid encodes anantigen and/or a non-vaccinogenic therapeutically active polypeptide,wherein the said antigen is capable of eliciting an immune response,preferably protective immune response, in a human or animal subject,and/or the said non-vaccinogenic therapeutically active polypeptide iscapable of producing a therapeutic effect in a human or animal subject.

WO 97/14806 further specifically discloses co-expression of antigenswith immune response stimulatory molecules, such as, e.g., interleukins,e.g., IL-2 or IL-6, by bacteria. Accordingly, freeze-dried bacteria ofthe invention for such co-expression are also contemplated.

In a further preferred embodiment, the open reading frame according tothe invention further comprises a sequence encoding a secretion signalin phase with a polypeptide encoded by the ORF. This advantageouslyallows for secretion of the expressed polypeptide from the host cell andthereby may facilitate, e.g., isolation or delivery.

Typically, a secretion signal sequence represents an about 16 to about35 amino acid segment, usually containing hydrophobic amino acids thatbecome embedded in the lipid bilayer membrane, and thereby allow for thesecretion of an accompanying protein or peptide sequence from the hostcell, and which usually is cleaved from that protein or peptide.Preferably, the secretion signal sequence may be so-active in a hostcell intended for use with the nucleic acid comprising the said signalsequence, e.g., a bacterial host cell, preferably a lactic acidbacterium, more preferably Lactococcus, even more preferably Lactococcuslactis.

Secretion signal sequences active in suitable host cells are known inthe art; exemplary Lactococcus signal sequences include those of usp45(see, U.S. Pat. No. 5,559,007) and others, see, e.g., Perez-Martinez etal. 1992 (Mol Gen Genet 234: 401-11); Sibakov et al. 1991 (Appl EnvironMicrobiol 57(2): 341-8). Preferably, the signal sequence is locatedbetween the promoter sequence and the ORF, i.e. the signal sequence islocated 3′ from the promoter sequence and precedes the ORF of thepolypeptide of interest. In a preferred embodiment, the signal sequenceencodes the amino acid sequence

MKKKIISAIL MSTVILSAAA PLSGVYA (usp45).

In a further aspect, the freeze-dried bacterium of the inventioncomprises a vector containing a recombinant nucleic acid.

As used herein, “vector” refers to a nucleic acid molecule, typicallyDNA, to which nucleic acid fragments may be inserted and cloned, i.e.,propagated. Hence, a vector will typically contain one or more uniquerestriction sites, and may be capable of autonomous replication in adefined host or vehicle organism such that the cloned sequence isreproducible. Vectors may include, without limitation, plasmids,phagemids, bacteriophages, bacteriophage-derived vectors, PAC, BAC,linear nucleic acids, e.g., linear DNA, etc., as appropriate (see, e.g.,Sambrook et al., 1989; Ausubel 1992).

The recombinant nucleic acid or the vector of the invention may bepresent in the host cell extra-chromosomally, preferably autonomouslyreplicating using an own origin of replication, or may be integratedinto bacterial genomic DNA, e.g., bacterial chromosome, e.g.,Lactococcus chromosome. In the latter case, a single or multiple copiesof the said nucleic acid may be integrated, preferably a single copy;the integration may occur at a random site of the chromosome or, asdescribed above, at a predetermined site thereof, preferably at apredetermined site, such as, in a preferred non-limiting example, in thethyA locus of Lactococcus, e.g., Lactococcus lactis.

In a related aspect, the invention provides a method for themanufacturing of freeze-dried bacteria comprising one or more openreading frames within a recombinant nucleic acid within the freeze-driedbacteria to human or animal in need thereof, comprising administering tothe said human or animal a therapeutically effective amount of suchbacteria transformed with the said nucleic acid.

The animal may preferably be a mammal, such as, e.g., domestic animals,farm animals, zoo animals, sport animals, pet and experimental animalssuch as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle,cows; primates such as apes, monkeys, orang-utans, and chimpanzees;canids such as dogs and wolves; felids such as cats, lions, and tigers;equids such as horses, donkeys, and zebras; food animals such as cows,pigs, and sheep; ungulates such as deer and giraffes; rodents such asmice, rats, hamsters and guinea pigs; and so on.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder. A “human or animal in need oftreatment” includes ones that would benefit from treatment of a givencondition.

The term “therapeutically effective amount” refers to an amount of atherapeutic substance or composition effective to treat a disease ordisorder in a subject, e.g., human or animal, i.e., to obtain a desiredlocal or systemic effect and performance. By means of example, atherapeutically effective amount of bacteria may comprise at least 1bacterium, or at least 10 bacteria, or at least 10² bacteria, or atleast 10³ bacteria, or at least 10⁴ bacteria, or at least 10⁵ bacteria,or at least 10⁶ bacteria, or at least 10⁷ bacteria, or at least 10⁸bacteria, or at least 10⁹, or at least 10¹⁰, or at least 10¹¹, or atleast 10¹², or at least 10¹³, or at least 10¹⁴, or at least 10¹⁵, ormore host cells, e.g., bacteria, e.g., in a single or repeated dose.

The freeze-dried cells of the present invention may be administeredalone or in combination with one or more active compounds. The lattercan be administered before, after or simultaneously with theadministration of the said freeze-dried cells.

A number of prior art disclosures on the delivery of antigens and/ortherapeutically active polypeptides exist, and it shall be appreciatedthat such disclosures may be further advantageously modified with thestrong promoters of the present invention. By means of example and notlimitation, bacterial delivery of trefoil peptides may be used to treatdiseases of the alimentary canal (see, e.g., WO 01/02570), delivery ofinterleukins in particular IL-10 for treating colitis (e.g., see WO00/23471), delivery of antigens as vaccines (e.g., WO 97/14806),delivery of GLP-2 and related analogs may be used to treat short boweldisease, Crohn's disease, osteoporosis and as adjuvant therapy duringcancer chemotherapy, etc. Further therapeutic applications areenvisioned using the freeze-dried cells of the invention.

Further non-limiting examples of the types of diseases treatable inhumans or animals by delivery of therapeutic polypeptides according tothe invention include, but are not limited to, e.g., inflammatory boweldiseases including Crohn's disease and ulcerative colitis (treatablewith, e.g., IL-Ira or IL-10 or trefoil peptides); autoimmune diseases,including but not limited to psoriasis, rheumatoid arthritis, lupuserythematosus (treatable with, e.g., IL-Ira or IL-10); neurologicaldisorders including, but not limited to Alzheimer's disease, Parkinson'sdisease and amyotrophic lateral sclerosis (treatable with, e.g., braindevated neurotropic factor and ciliary neurotropic factor); cancer(treatable with, e.g., IL-1, colony stimulating factors orinterferon-W); osteoporosis (treatable with, e.g., transforming growthfactorf3); diabetes (treatable with, e.g., insulin); cardiovasculardisease (treatable with, e.g., tissue plasminogen activator);atherosclerosis (treatable with, e.g., cytokines and cytokineantagonists); hemophilia (treatable with, e.g., clotting factors);degenerative liver disease (treatable with, e.g., hepatocyte growthfactor or interferon a); pulmonary diseases such as cystic fibrosis(treatable with, e.g., alpha antitrypsin); obesity; pathogen infections,e.g., viral or bacterial infections (treatable with any number of theabove-mentioned compositions or antigens); etc.

In a further aspect, the invention thus also provides a pharmaceuticalcomposition comprising the freeze-dried bacteria manufactured by theinvention, whether or not transformed with the nucleic acid and/or thevector described aboven.

Preferably, such formulation comprise a therapeutically effective amountof the freeze-dried bacteria manufactured by the invention and apharmaceutically acceptable carrier, i.e., one or more pharmaceuticallyacceptable carrier substances and/or additives, e.g., buffers, carriers,excipients, stabilisers, etc. The term “pharmaceutically acceptable” asused herein is consistent with the art and means compatible with theother ingredients of a pharmaceutical composition and not deleterious tothe recipient thereof. Freeze-dried bacteria lyophilized according tothe invention may be prepared in the form of capsules, tablets,granulates and powders, each of which may be administered by the oralroute.

Alternatively, freeze-dried bacteria lyophilized according to theinvention may be prepared as aqueous suspensions in suitable media, orlyophilized bacteria may be suspended in a suitable medium just prior touse.

For oral administration, gastroresistant oral dosage forms may beformulated, which dosage forms may also include compounds providingcontrolled release of the host cells and thereby provide controlledrelease of the desired protein encoded therein. For example, the oraldosage form (including tablets, pellets, granulates, powders) may becoated with a thin layer of excipient (usually polymers, cellulosicderivatives and/or lipophilic materials) that resists dissolution ordisruption in the stomach, but not in the intestine, thereby allowingtransit through the stomach in favour of disintegration, dissolution andabsorption in the intestine.

The oral dosage form may be designed to allow slow release of the hostcells and of the recombinant protein thereof, for instance as controlledrelease, sustained release, prolonged release, sustained action tabletsor capsules. These dosage forms usually contain conventional and wellknown excipients, such as lipophilic, polymeric, cellulosic, insoluble,swellable excipients. Controlled release formulations may also be usedfor any other delivery sites including intestinal, colon, bioadhesion orsublingual delivery (i.e., dental mucosal delivery) and bronchialdelivery. When the compositions of the invention are to be administeredrectally or vaginally, pharmaceutical formulations may includesuppositories and creams. In this instance, the host cells are suspendedin a mixture of common excipients also including lipids. Each of theaforementioned formulations are well known in the art and are described,for example, in the following references: Hansel et al. (1990,Pharmaceutical dosage forms and drug delivery systems, 5th edition,William and Wilkins); Chien 1992, Novel drug delivery system, 2ndedition, M. Dekker); Prescott et al. (1989, Novel drug delivery, J.Wiley & Sons); Cazzaniga et al., (1994, Oral delayed release system forcolonic specific delivery, Int. J. Pharm.i08:7′).

Preferably, an enema formulation may be used for rectal administration.The term “enema” is used to cover liquid preparations intended forrectal use. The enema may be usually supplied in single-dose containersand contains one or more active substances dissolved or dispersed inwater, glycerol or macrogols or other suitable solvents.

Thus, according the invention, in a preferred embodiment, recombinanthost cells encoding a desired gene may be administered to the animal orhuman via mucosal, e.g., an oral, nasal, rectal, vaginal or bronchialroute by any one of the state-of-the art formulations applicable to thespecific route. Dosages of host cells for administration will varydepending upon any number of factors including the type of bacteria andthe gene encoded thereby, the type and severity of the disease to betreated and the route of administration to be used.

Thus, precise dosages cannot be defined for each and every embodiment ofthe invention, but will be readily apparent to those skilled in the artonce armed with the present invention. The dosage could be anyhowdetermined on a case by case way by measuring the serum levelconcentrations of the recombinant protein after administration ofpredetermined numbers of cells, using well known methods, such as thoseknown as ELISA or Biacore (See examples). The analysis of the kineticprofile and half life of the delivered recombinant protein providessufficient information to allow the determination of an effective dosagerange for the transformed host cells.

In an embodiment, when the freeze-dried bacteria manufactured inaccordance with the inventions express an antigen, the invention maythus also provide a vaccine.

The term “vaccine” identifies a pharmaceutically acceptable compositionthat, when administered in an effective amount to an animal or humansubject, is capable of inducing antibodies to an immunogen comprised inthe vaccine and/or elicits protective immunity in the subject.

The vaccine of the invention would comprise the host cells transformedwith the nucleic acids or vectors of the invention and furtheroptionally an excipient. Such vaccines may also comprise an adjuvant,i.e., a compound or composition that enhances the immune response to anantigen. Adjuvants include, but are not limited to, complete Freund'sadjuvant, incomplete Freund's adjuvant, saponin, mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions,and potentially useful pharmaceutically acceptable human adjuvants suchas BCG (bacille Calmetle-Guerin) and Corynebacterium parvum.

The freeze-dried lactic acid bacteria of the invention can be used inthe food industry in general as a food additive or in particular as astarter culture, such as yoghurt starter cultures or cheese startercultures. Typically, such compositions comprise the bacteria in aconcentrated form including frozen, dried or freeze-dried concentratestypically having a concentration of viable cells which is at least10<5>CFU per gram of the composition, such as at least 10<6>CFU/gincluding at least 10<7>CFU/g, e.g. at least 10<8>CFU/g, e.g. at least10<10>CFU/g, such as at least 10<11>CFU/g, e.g. at least 10<12> of thecomposition. The composition may as further components containconventional additives including nutrients such as yeast extract, sugarsand vitamins.

The invention provides for a method to manufacture freeze-dried lacticacid bacteria that are useful for a variety of edible product componentsor ingredients such as milk including non-pasteurized (raw) milk, meat,flour dough, wine and plant materials, such as vegetables, fruits orfodder crops. As used herein, the term “milk” is intended to mean anytype of milk or milk component including e.g. cow's milk, human milk,buffalo milk, goat's milk, sheep's milk, dairy products made from suchmilk, or whey. The particular advantage of the freeze-dried lactic acidbacteria of the invention is the high level of viability and long-termstorage capacity. The starter culture is added in amounts which resultin a number of viable cells which is at least 10<3> colony forming units(CFU) per gram of the edible product starting materials, such as atleast 10<4>CFU/g including at least 10<5>CFU/g, such as at least10<6>CFU/g, e.g. at least 10<7>CFU/g, such as at least 10<8>CFU/g, e.gat least 10<9>CFU/g, such as at least 10<10>CFU/g, e.g. at least10<11>CFU/g, e.g. at least 10<12>/g of the edible product startingmaterials.

The invention also provides a freeze dried lactic acid bacteriumcomprising a combination of stabilizing compounds of the invention,which is in the form of a starter culture composition for the productionof a food product or an animal feed, or in the form of a culture for theproduction of an aroma.

The invention is further illustrated with examples that are not to beconsidered limiting.

Examples

This invention will be better understood by reference to theExperimental Details that follow, but those skilled in the art willreadily appreciate that these are only illustrative of the invention asdescribed more fully in the claims that follow thereafter. Otherembodiments will occur to the person skilled in the art in light ofthese examples, in particular embodiments comprising other lactic acidbacterium as Lactococcus species, such as a Lactobacillus species, aStreptococcus species, a Pediococcus species, a Bifidobacterium speciesand a Leuconostoc species and any to taxon (e.g., species, subspecies,strain) classified as belonging to such in the art.

Example 1: Stabilizing Effect of a Combination of Dextrane, SodiumGlutamate and a Polyol During Freeze-Drying and Unprotected Short-Term(24 Hours) Exposure to 25° C./35% RH

L. Lactis strain sAGX0037 was grown in a 5 L fermentor, washed twicewith purified water that contained 800 μM thymidine, and concentratedtenfold. The concentrated bacteria were mixed in a 1:1 volume ratio withthe cryoprotectant mixture (1 ml sample+1 ml cryoprotectant). Theresulting formulation was aliquotted to 35 vials in 2 ml volumes. Theentire process, from the bioreactor to the vial, took approximately 8hours, while the temperature was averaging 10° C. The variousformulations in their final concentration, i.e. including bacteria, areshown in Table 1. The vials were frozen in solid CO2 pellets until thesewere placed in the freeze-dryer.

TABLE 1 Composition of the cryoprotectant formulations after adding thecell suspension. The dry weight content of the cells in the formulatedcell suspension was 33 g/L or 3.3%. Total solid cryoprotectant matter infinal liquid formulation before freeze- Final composition drying Code(weight/volume [w/v]) (in % w/v) A 5% HES Hydroxy Ethyl Starch 12.5%  5% trehalose 2.5% Sodium Glutamate B 4% sorbitol 20% 4% dextrane 500 4%HES 4% trehalose 4% glycine C 7.5% HES 20% 10% sucrose 2.5% SodiumGlutamate D 7.5% mannitol 20.5%   7.5% Sodium Glutamate 3% glycine 2.5%dextrane 500 E 10% sucrose 10% F no excipients added

In order to test the survival of the freeze-dried cakes after exposureto environmental conditions (mimicking downstream formulation processes[e.g. capsule filling]), 3 vials were analysed on viable cell countimmediately after freeze-drying and after 24 hours of storage at 25° C.and 35% RH.

The results of the survival analysis for L. lactis strain sAGX0037,determined by viable cell count on the freeze-dried samples as well asthe samples exposed to air, indicated that a combination of a starchhydrolysate (dextrane 500), sodium glutamate and a polyol (e.g.mannitol) (as presented by Code D), protected the freeze-dried L. lactisbacteria upon unprotected storage for 24 hours at 25° C. and 35% RH.

Although combinations containing either sodium glutamate or sorbitol anddextrane, combined with well-known cryoprotectants such as trehalose andsucrose (formulations coded A, B and C) resulted in viable cell yieldsimmediately after freeze-drying that were comparable to the formulationcoded D (>1×10E+11 CFU/g), short-term exposure studies clearlydemonstrated that only formulation D, containing a combination of astarch hydrolysate and a glutamic acid salt and/or a polyol, protectedfreeze-dried L. lactis bacteria upon unprotected storage during 24 hoursat 25° C. and 35% RH.

Compared to sucrose formulation (coded E), known as a “golden standard”for stabilisation of freeze-dried LAB, the selected combination (D) issuperior upon storage, and is the only combination in this exampleresulting in a viable cell count >1×10E+11 CFU/g upon short-termexposure to 25° C./35% RH.

When no stabilisers were added to the bacteria, no survival of thebacteria was observed upon short-term exposure, as demonstrated in FIG.1, formulation coded F.

Example 2: Stabilizing Effect of a Combination of Dextrane, SodiumGlutamate and a Polyol During Freeze-Drying and Short-Term (24 Hours)Exposure to 25° C./35% RH

A pre-culture (100 ml) of L. lactis strain sAGX0037 was used for theinoculation of a 7 L Continuously Stirred Tank Reactor (CSTR),containing 5 L of M17c medium (composition listed in Table 2)

TABLE 2 Composition of M17c fermentation medium Component Quantity (for1 litre) Yeast extract 20 g KH₂PO₄ 2.0 g MgSO₄, 7H₂0 0.51 g Citric acidmonohydrate 0.49 g Glucose 55 g Thymidine (100 mM) 8 ml/L

The bioreactor was set to maintain temperature at 30° C. and pH to 7 (byaddition of 5M NH4OH). The agitation speed was set at 200 rpm. Thefermentation was terminated when the glucose consumption was completedby cooling the fermentor to 4′C. An ‘end of fermentation’ sample wastaken and used for dry cell weight (DCW) determination. Once thefermentation was terminated, 3.5 L of the fermentation broth wasconcentrated and washed by ultrafiltration/diafiltration using a 1400cm2 500 kDa hollow fibre filter.

When the total amount of 3.5 L broth was concentrated approximately10-fold, the 5 L bulk bottle was replaced with a bottle containingpurified water and used for diafiltration. During diafiltration, thelactate concentration was monitored by analysis of the permeate.Diafiltration was terminated once the lactate concentration reached 5-10g/L.

Directly after concentration and diafiltration, the bacterial cellsuspensions were divided in 13 portions, to which differentcryoprotectants were added (listed in Table 3). After mixing thecryoprotectants with the cell suspensions, each mixture was aliquottedover 25 vials (2 ml end volume) and freeze-dried.

TABLE 3 Composition of the cryoprotectant formulations after adding thecell suspension. The dry weight content of the cells in the formulatedcell suspension was 70 g/L Total solid Cryoprotectant Composition matterin Volume cell Volume Formulation cryoprotectant final liquidformulation (% Suspension Cryoprotectant code Solution (w/v) w/v) (ml)Solution (ml) A1 10% HES 12.5%   26 26 10% trehalose 5% Sodium GlutamateA2 5% HES 10% 26 26 10% trehalose 5% Sodium Glutamate B1 8% sorbitol 20%26 26 8% dextrane 500 8% HES 8% trehalose 8% glycine C1 15% HES 20% 2626 20% sucrose 5% Sodium Glutamate C2 15% HES 12.5%   26 26 15% sucrose5% Sodium Glutamate D1 15% mannitol 20.5%   26 26 15% Sorbitol 5% SodiumGlutamate 6% glycine 5% dextrane 500 D2 7% mannitol 12% 26 26 7%Sorbitol 5% Sodium Glutamate 5% dextrane 500 E1 50% sucrose 10% 41.610.4 F2 40% sodium glutamate  5% 40 6.29

In order to test bacterial survival on the freeze-dried cakes afterexposure to environmental conditions (mimicking downstream formulationprocesses [e.g. capsule filling]), 3 vials were analysed for viable cellcount immediately after freeze-drying and after 24 hours of storage at25° C. and 35% RH.

The results of the survival analysis for L. lactis strain sAGX0037,determined by viable cell count on the lyophilized samples as well asthe samples exposed to air, indicated that a combination of a starchhydrolysate (dextrane 500), a polyol (e.g. mannitol and/or sorbitol) andsodium glutamate (as presented by code D1), yielded high viable cellscount immediately after freeze-drying (e.g. >3 E+11 CFU/g) andstabilised the freeze-dried L. lactis bacteria upon unprotected storagefor 24 hours at 25° C. and 35% RH, resulting in high viable cell yields(>2 E+11 CFU/g).

Although the formulations coded A1, A2, B1, C1 and C2 (combinationscontaining either sodium glutamate or sorbitol and dextrane, combinedwith well-known cryoprotectants such as trehalose and sucrose) resultedin viable cell yields immediately after freeze-drying that werecomparable to the formulation coded D1, short-term exposure studiesclearly demonstrated that formulations D1 and D2 (containing a mixtureof at least a starch hydrolysate [dextrane 500], a polyol [e.g. mannitoland/or sorbitol] and sodium glutamate) stabilized the freeze-dried L.lactis bacteria upon unprotected storage during 24 hours at 25° C. and35% RH.

Compared to sucrose formulation (coded E1), a “golden standard” forstabilisation of freeze-dried LAB, the combination of 3 cryoprotectantsis clearly superior upon storage, and is the only combination ofstabilisers in this example that resulted in a viable cellcount >2×10E+11 CFU/g upon short-term exposure to 25° C./35% RH.

When sodium glutamate alone was added to the bacteria, no survival ofthe bacteria was observed upon short-term exposure, as demonstrated inFIG. 2, formulation coded F2.

Example 3: Stabilizing Effect of a Combination of Dextrane, SodiumGlutamate and Sorbitol During Freeze-Drying and Unprotected Short-Term(4 and 24 Hours) Exposure to 25° C./35% RH

A pre-culture of L. lactis strain sAGX0037 (100 ml) was used forinoculation of a 7 L CSTR containing 5 L of M17c medium (see Table 2above). The bioreactor was set to maintain temperature at 30° C. and pHto 7 (by addition of 5M NH4OH). The agitation speed was set to 200 rpm.The fermentation was terminated when the glucose concentration haddropped below 0.5 g/L, by cooling the fermentor to 4° C. An ‘end offermentation’ sample was taken and used for DCW determination. Once thefermentation was terminated, 3.5 L of the fermentation broth wasconcentrated and washed by ultrafiltration/diafiltration using a 1400cm2 500 kDa hollow fibre filter.

When the total amount of 3.5 L broth was concentrated approximately10-fold, the 5 L bulk bottle was replaced with a bottle containingpurified water, and used for diafiltration. During diafiltration, thelactate concentration was monitored by analysis of the permeate.Diafiltration was terminated once the lactate concentration reached 5-10g/L.

The bacteria suspensions were mixed with different cryoprotectants, thecomposition of which is described in Table 4. After mixing thecryoprotectants with the suspensions, each mixture was aliquotted indifferent freeze-drying containers (55 ml aliquots) under asepticconditions. After aliquotting, the containers were placed on a flatplate into a −70° C. freezer until freeze-drying. Two secondary dryingtemperatures were evaluated during the freeze-drying process: 25° C. and35° C. shelf temperature.

TABLE 4 Composition of the cryoprotectant formulations after adding thecell suspension (dry weight of 70 g/L). Final Cryoprotectant Mixturecomposition Volume in Composition 5 × Volume cell Cryoprotectant liquidformulation Cryoprotectant concentrated Suspension bulk solution beforefreeze-drying code bulk solution (w/v) (ml) (ml) (% w/v) Z4 20%Na-glutamate 184 46 4% Na-glutamate 25° C./35° C. 10% dextrane 500 2%dextrane 500 10% sorbitol 2% sorbitol

The results of the viability analysis for L. lactis strain sAGX0037(FIG. 3), determined by viable cell count on the lyophilized samples aswell as on the samples exposed to air, indicated that a combination of astarch hydrolysate (dextrane 500), a polyol (e.g. mannitol and/orsorbitol) and sodium glutamate, yielded high viable cell countsimmediately after freeze-drying (e.g. >6 E+11 CFU/g) and stabilised thefreeze-dried L. lactis bacteria upon unprotected exposure to 25° C. and35% RH for 4 hours and 24 hours. High viable cell stability was observedafter this exposure test, resulting in viable cell yields of >6 E+11CFU/g after 4 hours and >5 E+11 CFU/g after 24 hours of exposurerespectively.

TABLE 4A Effect of cryoprotectant formulation on survival of L. lactisafter freeze drying Viable cell cell counting count CFU/ml Endcomposition CFU/ml after freeze Cryoprotectant before drying and Samplemixture freeze drying reconstitution Survival % Z4 4% Na-glutamate1.6E+11 1.1E+11 69% 25° C. 2% dextran 500 2% sorbitol

Example 4: Stabilizing Effect of a Combination of Dextrin, SodiumGlutamate and Sorbitol During Freeze-Drying and Long-Term Storage atDifferent Storage Conditions

After a 200 L scale industrial fermentation of L. lactis strainsAGX0037, the accumulated biomass was concentrated and washed withpurified water by ultrafiltration/diafiltration respectively.Diafiltration was stopped once the lactate concentration dropped below 5g/L (55 mmol/L). During ultrafiltration and diafiltration, the vesseljacket was continuously water-cooled to 4° C.

In view of the subsequent lyophilization step, stabilization of thebacteria was ensured by addition of a selected cryoprotectant solutionto the biomass resulting from the UF/DF step. The final cryoprotectantsolution consisted of a starch hydrolysate (dextrin from maize starch),sodium glutamate and a polyol (sorbitol), as described in Table 5.

TABLE 5 Composition of the cryoprotectant formulation Component WeightDextrin (from maize starch) 100 g = 10% w/v Sorbitol 100 g = 10% w/vSodium glutamate 200 g = 20% w/v Water For Injections QSP 1 L

The required weight of the added cryoprotectant solution wasapproximately 17.0 kg cell suspension and 4.8 kg cryoprotectantsolution, resulting in an approximate final formulation weight of 21.8kg formulated cell suspension. The formulation was dispatched intosuitable bulk lyophilization trays and freeze-dried under validated andmonitored conditions. The trays were loaded onto the shelves of thefreeze-dryer and subsequently frozen to −50° C. The total freezing timewas approximately 9 hours.

After the freezing step, the chamber pressure was decreased and primarydrying was started by increasing the shelf temperature in multiple rampsteps to −22° C., −10° C., 20° C., 25° C. and a final shelf temperatureof 35° C. At the end of primary drying, a pressure rise test wasperformed to determine the end of the primary drying phase. No pressurerise occurred, and the freeze-drying process was continued by asecondary drying phase. The total time of the freeze-drying process wasapproximately 93 hours.

At the end of the freeze-drying cycle, the chamber was pressurized withdry, sterile-filtered nitrogen gas (filtered on a 0.22 μm pore sizemembrane). The trays were unloaded and packed into vapour-impermeablealuminium (Alu) foil pouches, at 18-26° C. and 30-70% RH respectively.

Then, the freeze-dried cakes were equilibrated for approximately onehour in a Class 100.000 production room at controlled temperature(19-23° C.) and humidity (<20% RH), and subsequently powdered by manualgrinding (in PE bags) of the lyophilized cakes, followed by sieving (410μm). After sieving, the powder was homogenized manually (in PE bags) andsamples were taken from the resulting final freeze-dried bulk DrugSubstance (DS, containing L. lactis sAGX0037 bacteria andcryoprotectants), for analysis and stability testing.

Samples were packed per 500 mg in PET/Alu bags and stored at −20±5° C.,at 5±3° C. and at 25±2° C., 60±5% RH. The samples were monitored during12 months. As demonstrated in FIG. 4, no significant decrease in viablecell count was observed on the powdered, freeze-dried bacteria stored at−20° C. and 5° C., and >90% of the initial viable cell count waspreserved during 1 year of storage, resulting in very high CFUconcentrations of up to >5×10E+11 CFU/g. At 25° C., 60% RH, only aslight decrease in viable cell count was observed, resulting in high CFUconcentrations of up to >3×10E+11 CFU/g after 1 year of storage at 25°C./60% RH.

These data clearly demonstrate that the cryoprotectant combination of astarch hydrolysate (e.g., dextrin from maize starch), sodium glutamateand a polyol (e.g. sorbitol) leads to a stable freeze-dried LAB powder,assuring long-term stability and survival of viable bacteria.

REFERENCES

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1.-12. (canceled)
 13. A composition comprising: (a) a biomass of freezedried Lactococcus lactis bacteria; and (b) stabilizing compounds,wherein a ratio of the biomass to the stabilizing compounds is no morethan 1:1 (dry weight biomass to dry weight stabilizing compounds),wherein the stabilizing compounds consist essentially of: (i) a starchhydrolysate, wherein the starch hydrolysate is selected from dextran anddextrin, (ii) a glutamic acid salt, and (iii) a polyol, wherein thepolyol is selected from sorbitol and mannitol, and wherein thecomposition is free of milk.
 14. The composition according to claim 13,wherein prior to freeze drying: the amount of starch hydrolysate rangesfrom about 2.0% to about 10% (w/v); the amount of glutamic acid saltranges from about 2.0% to about 10% (w/v); and the amount of polyolranges from about 5.0 to about 30% (w/v).
 15. The composition accordingto claim 13, wherein the glutamic acid salt is sodium glutamate.
 16. Thecomposition according to claim 13, wherein the starch hydrolysate isdextran.
 17. A method for freeze drying Lactococcus lactis bacteriacomprising: (a) providing a bacterial biomass comprising Lactococcuslactis bacteria; (b) mixing the biomass with a combination ofstabilizing compounds in a ratio of no more than 1:1 (dry weight biomassto dry weight stabilizing compounds) to form a composition, wherein thecombination of stabilizing compounds consist essentially of: (1) astarch hydrolysate, wherein the starch hydrolysate is selected fromdextran and dextrin, (2) a glutamic acid salt, and (3) a polyol, whereinthe polyol is selected from sorbitol and mannitol; and (c) freeze dryingthe composition, wherein the biomass is free of milk.
 18. Thecomposition according to claim 13, wherein the Lactococcus lactisbacterium comprises one or more recombinant nucleic acids which areheterologous to the Lactococcus lactis bacterium.
 19. A culture orstarter culture composition comprising the freeze dried compositionaccording to claim
 13. 20. A medicament comprising the freeze driedcomposition according to claim
 13. 21. A food ingredient comprising thefreeze dried composition according to claim
 13. 22. A food product oranimal feed comprising the culture or starter culture compositionaccording to claim
 17. 23. A composition comprising: (a) a biomass offreeze dried Lactococcus lactis bacteria; and (b) stabilizing compounds,wherein a ratio of the biomass to the stabilizing compounds is no morethan 1:1 (dry weight biomass to dry weight stabilizing compounds),wherein the stabilizing compounds consist essentially of: (i) a starchhydrolysate selected from dextran and dextrin, wherein the amount of thestarch hydrolysate ranges from about 5% to about 10% (w/v); (ii) sodiumglutamate, wherein the amount of the sodium glutamate ranges from about5.0% to about 10% (w/v); and (iii) a polyol selected from sorbitol andmannitol, wherein the amount of the polyol ranges from about 10% toabout 20% (w/v), and wherein the composition is free of milk.
 24. Thecomposition according to claim 23, wherein the starch hydrolysate isdextrin and the polyol is sorbitol.
 25. The composition according toclaim 23, wherein the freeze dried composition is free of animal-derivedcompounds.
 26. The composition according to claim 13, wherein theLactococcus lactis bacterium was cultured from a medium lacking milk.27. The composition according to claim 13, wherein the composition isfree of animal-derived compounds.
 28. The composition according to claim13, wherein the Lactococcus lactis bacterium in the composition retainsat least 50% viability after 12 months when stored at −20° C. or 5° C.29. The composition according to claim 13, wherein the Lactococcuslactis bacterium in the composition retains at least 80% viability after12 months when stored at −20° C. or 5° C.
 30. The composition accordingto claim 13, wherein the starch hydrolysate is dextrin and the polyol issorbitol.
 31. The composition according to claim 13, wherein theLactococcus lactis bacterium is genetically modified.